Separation of many peptides present in tissue which often are structurally very similar requires the highest possible resolution. High pressure liquid chromatography (HPLC) probably has the highest resolution for peptides of any method available today, but incomplete understanding of parameters affecting resolution and retention has hindered the application of the method. A number of factors controlling retention of peptides in HPLC were evaluated in this study. These results will aid in rapid specific isolation of peptides for quantitative analysis.